Studies in previous years have shown that high affinity anti-yeast iso-1 cytochrome c monoclonal antibodies (mAb) 2-96-12 and 4-74-6 have closely related epitopes. However, while the specificity of 4-74-6 is very stringent, 2-96-12 cross reacts with many evolutionarily related cyts. c. To obtain a clue to the molecular basis of this phenomenon, we have carried out molecular modeling of Fv fragments of 2-96-12, 4-74-6 and another anti-iso-1 mAb 4-128-6 as a control. A Fv fragment consist of the variable domains V-L and V-H of the light and heavy chains, respectively of immunoglobins. Previously determined cDNA sequences were used to deduce the amino acid sequences. The exception is that 8 amino-terminal residues of V-H and V-L were not determined for 4-74-6 and 4-128-6 and that 8 amino-terminal residues of V-L of 2-96-12 were deduced from the mRNA sequence. The results show the following: The presentation of amino acids in the combining site is distinctly different in 2-96-12 and 4-74-6, especially with respect to aromatic residues. Arg 95 H in 2-96-12 and Arg 91L in 4-74-6 conspicuously project their side chains toward the central region of the combining site or its vicinity. These side chain positions are supported by comparative studies of known structures of antibody Fab fragments. A substitution test of the Arg 95 H in previous years is also compastible with the model. Epitope analysis by docking of the antigen to the Fv models was also performed. Previous immunological studies and present results of docking, taken together, are compatible with the idea that iso-1 Asp 60 and Glu 61 would be positioned closely to the Arg 95 H in 2-96-12 and the Arg 91 L in 4-74-6 in the antigen -antibody complex. Thus, we propose that the distinct difference in stringency of the antigen recognition between 2-96-12 and 4-74-6 would be related to the distinctly different presentation of amino acids in the combining site despite similarity in the epitopes.